COMBINATORIAL CIS-ACTING ELEMENTS CONTROL TISSUE-SPECIFIC ACTIVATION OF THE CARDIAC TROPONIN-I GENE IN-VITRO AND IN-VIVO

The cardiac troponin I gene is one of the few sarcomeric protein genes exclusively expressed in cardiac muscle. We show here that this speci ficity is controlled by a proximal promoter (-230/+16) in transfected cardiac cells in culture, in the adult hearts, and in transgenic anima ls. Functional analysis indicates that MEF2/Oct-1, Spl, and GATA regul atory elements are required for optimal gene activation because select ive mutations produce weak or inactive promoters. MEF2 and Oct-1 trans cription factors bind to the same APT-rich element. A mutation that bl ocks this binding markedly reduces gene activation in vivo and in vitr o, and overexpression of MEF2A, MEF2C, and MEF2D in noncardiac cells t ransactivates the cardiac troponin I promoter. Disruption of these ele ments inactivates the cardiac troponin I promoter in cultured cardiac cells but has a less important role in transfected adult heart. Moreov er, nuclear extracts from an almost pure population of adult cardiac c ells contain much lower levels of GATA binding activity compared with fetal cardiac cells. These findings point to a differential role of GA TA factors in the maintenance of gene expression in the adult heart as compared with the activation of cardiac genes in fetal cardiomyocytes , Overexpression of GATA family members transactivates the cardiac tro ponin I promoter, and GATA-5 and GATA-6 are stronger transactivators t han GATA-4, a property apparently unique to the cardiac troponin I pro moter. Transgenic mice carrying the -230/+126 base pair promoter expre ss beta-galactosidase reporter gene in the heart both at early stages of cardiogenesis and in the adult animals. These results indicate that the ability of the cardiac troponin I proximal promoter to target exp ression of a downstream gene in the heart is also maintained when the transgene is integrated into the genome.